Hepatitis B virus mutants and fulminant hepatitis B: Fitness plus phenotype

Editorial

Hepatitis B Virus Mutants and Fulminant Hepatitis B: Fitness Plus Phenotype

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The human hepatitis B virus (HBV) is the smallest known double-stranded DNA virus of man that replicates its genome through a novel mechanism of reverse transcription using the RNA-replicative intermediate and the viral pregenomic RNA.1 Viral pregenomic RNA is synthesized from molecules of covalently closed circular (ccc) DNA, the major transcriptional template of the virus, functioning as a viral minichromosome within the nucleus of infected cells.2 The reverse transcriptase of HBV lacks a conventional proofreading function, which is found in higher-order polymerases and, not surprisingly then, HBV exhibits a mutation rate more than 10-fold higher than other DNA viruses. The rates for nucleotide substitutions vary depending on the stage of disease. The natural evolutionary rate for the HBV genome in chronic hepatitis B is approximately 1.4 to 3.2 ⫻ 10⫺5 substitutions/site/year,3 whereas in the liver transplantation setting, it is almost 100-fold higher.4 Longitudinal studies have shown that these HBV mutations are not distributed evenly over the entire genome but rather tend to cluster into mutational patterns in particular parts of the viral DNA; in particular, the basal core promoter (BCP), the precore region and the “a” determinant of the viral envelope.5 Importantly, the type and number of mutations that accumulate in the HBV genome over time have been shown to be a marker of the duration and/or severity of the liver disease, or the type and intensity of the immune response.5 The outcome of HBV infection depends on the interplay between the virus, the hepatocyte, and the host’s immune response.6 Under normal circumstances, HBV is not cytopathic and liver damage is the result of the host’s immune response targeting infected hepatocytes. However, during special or unusual circumstances, HBV appears to be directly cytopathic for hepatocytes often causing unique histopathologic conditions such as fibrosing cholestatic hepatitis, which, before the advent of specific antiviral therapy,7 was invariably fatal. The molecular virologic and cellular basis for fulminant hepatitis B (FHB) has been the focus of intensive investigation, but to date the results have been controversial. In the report by Kalinina et al. in this issue of HEPATOLOGY, the investigators, have provided the first evidence, using in vitro

Abbreviations: HBV, hepatitis B virus; cccDNA, covalently closed circular DNA; BCP, basal core promoter; FHB, fulminant hepatitis B; HBeAg, hepatitis B e antigen; DHBV, duck hepatitis B virus; HBsAg, hepatitis B surface antigen; LMV, lamivudine. From Research & Molecular Development, VIDRL, North Melbourne, Victoria, Australia. Received June 6, 2001; accepted June 7, 2001. Address reprint requests to: Stephen Locarnini, M.D., Research & Molecular Development, VIDRL, 10 Wreckyn Street, North Melbourne, Victoria, 3051, Australia. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3402-0029$35.00/0 doi:10.1053/jhep.2001.26764

functional studies that mutations in the S gene of the HBV isolated at the time of FHB had caused a severe defect in viral particle secretion. Earlier studies from this group had shown that the infectious clones from the same samples exhibited enhanced replication levels.8 The enhanced replication and the severe defect in viral particle secretion most likely contributed to the fulminant clinical course of recurrent HBV infection posttransplantation. The patient was also hepatitis B e antigen (HBeAg) negative. REGULATION OF HEPADNAVIRAL cccDNA: ROLE OF THE VIRAL ENVELOPE

Virus persistence in infected cells during chronic infection depends on the maintenance of the HBV cccDNA pool.9 This pool of transcriptional templates appears to be very stable and studies by Summers et al. using the duck hepatitis B virus (DHBV), have shown that the level of viral cccDNA is regulated by the pre-S envelope proteins via negative feedback.10 Thus, early in infection when there are low amounts of surface envelope proteins, mature nucleocapsids are shunted into the nucleus whereby the newly synthesized viral genome is converted into cccDNA. The number of cccDNA molecules increases as infection progresses, and this leads to an increase in viral RNA synthesis. The viral envelope proteins are subsequently produced at high levels and exert a negative feedback on cccDNA amplification by redirecting the nucleocapsids into the pathway for virion formation. Failure in the regulation of the cccDNA copy number by the envelope protein has been shown to result in hepatocyte death.11 More recently, Lenhoff et al.12 generated a cytopathic mutant of DHBV, G133E in the preS protein of DHBV. Inoculation of this mutant into susceptible ducklings resulted in enhanced viral replication, increased the pool of viral cccDNA, and caused hepatocyte destruction. The liver damage caused by G133E DHBV subsided over time resulting in mild chronic hepatitis, similar to that observed in wild-type virus-infected birds, and coincided with a reduction of viral replication to wild-type virus levels in the liver. At least one noncytopathic revertant was identified from the serum of G133E-infected birds after recovery,12 indicating that acute liver injury can result from infection with a cytopathic hepadnavirus, but such viruses may be rapidly replaced by noncytopathic variants during persistent infection. In other words, these cytopathic viruses are not as replication fit as the wild-type in the context of persistent infection. The three envelope proteins of HBV are the large (L), medium (M), and small (S) and are derived from the same single open reading frame encoding the PreS1/ PreS2 and S genes.5 The envelope proteins have a common carboxyl terminus, but use different translational start codons. The proteins are secreted as subviral particles that contain no HBV DNA and are also involved directly in forming the viral envelope for virion assembly. The large envelope protein is also myristylated13

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and contains an endoplasmic reticulum retention signal sequence at its N terminus.14 All three HBV envelope proteins share a common N-linked glycosylation site (Asn X Thr) located within S at residue 146 (Asn-146). The M protein has an additional glycosylation site at amino acid 4 (Asn-4).15 The HBIG-vaccine escape mutant G145R is located adjacent to the conserved N-glycosylation site at codon 146. However, previous site-directed mutagenesis experiments have shown that this glycosylation site is not essential for effective virion secretion, whereas the glycosylation site in the M protein at codon 4 is.15 Interestingly, the report by Kalinina et al. showed that the G145R mutant alone inhibited virion secretion by 30% compared with wild-type levels. Overexpression of the large envelope protein can block the secretion of the subviral particles as well as virions and can lead to the formation of ground glass hepatocytes in the liver.16 Mutations in the S promoter have been observed in patients with severe HBV recurrence post–liver transplantation.17 Experimentally, these mutations in the CAAT element of the S promoter have been shown to cause a reduction in the S protein level and retention of the L protein intracellularly, with an adverse effect on virion assembly.18 Taken together mutations in the PreS1/PreS2/S genes must be considered in the context of how they affect the complex regulation and feedback mechanisms of HBV RNA and protein synthesis, virion formation, and intranuclear cccDNA levels. This balance is essential for efficient virus replication and maintenance of the noncytopathic virus-cell relationship. When the balance is disrupted by the emergence of particular HBV mutants with altered phenotype, the changed virus-cell relationship can cause hepatocyte necrosis and lead to the development of FHB. With this in mind, the isolation of HBV with unique mutation(s) from cases(s) of FHB is not enough by itself to establish virulence and pathogenicity. Functional studies characterizing the new viral phenotype and the replication competence of the HBV mutant are essential to link genotype with phenotype and derive an outcome measurement of viral replication fitness. HBV MUTANTS ASSOCIATED WITH FULMINANT HEPATITIS

Various viral mutations have been implicated in the etiology and pathogenesis of FHB, and in the majority of cases, HBV mutations that effect HBeAg expression have been detected.19,20 These HBV mutations either alter the transcription of HBeAg and are located in the BCP at A1762T and G1764A, or truncate the translated protein by the introduction of a stop codon (i.e., precore mutations at G1896A). Other mutations, deletions, and insertions within the promoter regions, that alter or create transcription factor binding sites have also been reported.20,21 Against this association, the same BCP mutations at A1762T and G1764A and the precore mutation G1896A have also been found in patients with chronic hepatitis B, as well as asymptomatic HBV infection.22 In addition, a number of studies have determined that there is a low prevalence of these mutations in cases of FHB,23 suggesting that there may be other factors involved. As well as mutations affecting HBeAg production, other mutations in the HBV genome associated with FHB have included HBV variants encoding changes in the core protein,24 the PreS2 gene,25 and S gene including the HBIG-vaccine escape mutations at G145R in conjunction with an insertion in the “a” determinant between amino acids 122 and 123 of the viral envelope.26 Several of the mutations in the major hydro-

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philic loop reported by Kalinina et al. are previously recognized vaccine escape and/or HBIG escape mutations.27,28 In the study by Ghany et al.,27 the escape mutations selected after HBIG treatment tended to cluster around codons 40-45, 114122, and 198-208. The region around 44-49 is within the major histocompatibility class I-restricted T-cell epitope of hepatitis B surface antigen (HBsAg). One of the patients in the study by Ghany et al.27 had a mutation profile that was similar to that found by Kalinina et al. (i.e., I/V40N, G44E, L49R, G145R, S204R). The secretion defect was not noted by Ghany et al.,27 but this was a clinical study. Kalinina et al. showed that their M5 clone encoding G145R, S204R, and L205V had a secretion defect of approximately 60% compared with wildtype and proposed that the basic arginine residues may be important for retention within the endoplasmic reticulum. The G145R mutation also results in a concomitant change in the HBV reverse transcriptase region of the polymerase at codon rtR/W143Q.29 This codon is located upstream from the conserved B domain found in RNA-dependent polymerase proteins. With the advent of antiviral treatment and prophylaxis protocols using both HBIG and nucleoside analogues in the transplantation setting, new HBV mutants have been selected with changes in the polymerase as well as the envelope protein. FHB has been reported after nucleoside analogue therapy alone or in combination with HBIG therapy.30-32 These HBV variants encoded the lamivudine-resistant changes in the reverse transcriptase region at rtM204I/V ⫹/⫺ rtL180M (W196S/L, I195M in HBsAg) as well as other changes in the genome. Bock et al.30 reported on a series of HBV mutants with HBsAg changes in the “a” determinant at either P120T or G145R, which also contained lamivudineresistant mutations. These “double mutants” replicated to a greater extent in vitro in the presence of lamivudine (LMV) than in the absence of LMV, an extraordinary finding. The entire HBV genome from the dominant quasispecies has been sequenced in a number of published reports. Thus, the unique genetic framework of the predominant HBV quasispecies with multiple mutations within a gene or genes associated with FHB have been studied.8,20,25,33 These more recent studies, including the report by Kalinina et al., suggest that multiple mutations throughout the genome may contribute to (1) an increase in viral replication fitness, (2) a change in viral gene expression, or (3) an alteration of cytotoxic T lymphocyte epitopes. These virologic markers either alone or in various combinations and the interplay between the virus and the host are clearly important in the development of FHB. FUNCTIONAL STUDIES: PHENOTYPE ASSESSMENT AND REPLICATION FITNESS

A review of the functional studies published to date reveals conflicting data with respect to the BCP and precore mutations and their effect on replication fitness, i.e., yield phenotype. The BCP mutations when engineered into an infectious clone were associated with an increase in virion production.34,35 Other mutations that created novel transcription factor binding sites in promoter or enhancer regions also had a high replication yield phenotype.30 However, other groups have shown that while the BCP mutations were associated with reduced HBeAg production, there was no significant effect on the amount of intracellular preS1/PreS2/S proteins or PreS/S messenger RNA transcripts.36 Importantly, the amount of virions released was similar to that of wild-type.

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Sterneck et al.8 have shown that HBV with a high replication phenotype is not by itself a prerequisite for FHB. These investigators amplified and cloned the entire genomes from 8 patients but found clones from only one patient from whom the clone had a high replication phenotype. This clone was the focus of the study in the follow-up paper of Kalinina et al. Interestingly, as shown by Kalinina et al., a number of different mutations may result in the same viral phenotype, and so it appears that for HBV at least, there is no single mutation that is a virulence marker for FHB. Many aspects of HBV research have been hampered by the lack of convenient and reliable in vitro cell culture systems that are capable of supporting complete cycles of HBV replication. This includes meaningful measurements of viral fitness. Current assays are based on transfection of an individual HBV complementary DNA construct into appropriate cells, and the yield or amount of HBV DNA detected by Southern blot after several days in culture in the absence of any selection pressure are then interpreted as measures of replicative fitness. These assays lack standardization, are often unable to detect, small but significant variations in the viral replication burst size, and are associated with high inter-/intra-assay variability. These assays can be used to measure changes in HBV phenotype as outlined above, but are unable to reliably discriminate different HBVs based on relative replicative fitness. This is best addressed by using in vitro coinfection competition assays similar to those developed for the human immunodeficiency virus.37,38 In these assays, by the process of Darwinian competition, there is the selection of the “fittest” virus, which becomes the predominant species after replication following coinfection. This can now be reliably done using the recently developed recombinant HBV baculovirus system,39 which has been successfully adapted to the study of a number of HBV mutants in vitro.40 The earlier observation that the lamivudine-resistant HBV mutants were replication impaired41 was always difficult to reconcile with the fact that these viruses become the dominant viral species after only a few months of antiviral treatment and were often associated with exacerbation of liver disease in chronically infected patients42 and were even the cause of graft failure in transplant recipients.32 Clearly, there are differences in viral replication between mutant and wild-type HBV that play an important role in the etiology of patients presenting with FHB. CONCLUSIONS

A number of factors now need to be considered in defining the virologic basis of FHB. Mutations detected during FHB should be investigated in the context of the entire HBV genomic framework. The introduction of specific mutations into an infectious clone for in vitro functional studies should provide an indication for the role of particular mutation(s) on viral replication. However, these studies are not necessarily definitive as they overlook the effect of multiple mutations found in viral genomes and their subsequent effect on pathogenesis. Preferably, further in vitro phenotypic studies are required to determine the effect of the mutations on all aspects of viral replication including transcription, translation, assembly, and release. Next, competitive coinfection experiments using mixtures of mutant and wild-type HBV should be used to determine the relative replication fitness of each HBV strain. Finally, animal models of HBV such as the tree shrew species Tupaia belangeri should be pursued to examine potential pathogenicity in vivo.43 In this way, the accurate measure-

HEPATOLOGY August 2001

ment of viral fitness coupled with identification of novel viral phenotypes should permit new insights into the pathogenesis of FHB caused by mutant HBV. ANGELINE BARTHOLOMEUSZ, PH.D. STEPHEN LOCARNINI, M.D. Research and Molecular Development VIDRL North Melbourne, Victoria, Australia REFERENCES 1. Summers J, Mason WS. Replication of the genome of a hepatitis B–like virus by reverse transcription of an RNA intermediate. Cell 1982;29:403415. 2. Bock CT, Schranz P, Schroder CH, Zentgraf H. Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes 1994;8:215-229. 3. Okamoto H, Imai M, Kametani M, Nakamura T, Mayumi M. Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission. Jpn J Exp Med 1987; 57:231-236. 4. Sterneck M, Gunther S, Gerlach J, Naoumov NV, Santantonio T, Fischer L, Rogiers X, et al. Hepatitis B virus sequence changes evolving in liver transplant recipients with fulminant hepatitis. J Hepatol 1997;26:754764. 5. Gunther S, Fischer L, Pult I, Sterneck M, Will H. Naturally occurring variants of hepatitis B virus. Adv Virus Res 1999;52:25-137. 6. Chisari F, Ferrari C. Hepatitis B virus immunopathogenesis. Ann Rev Immunol 1995;13:29-60. 7. Angus P, Richards M, Bowden S, Ireton J, Sinclair R, Jones B, Locarnini S. Combination chemotherapy controls severe post-liver transplant recurrence of hepatitis B virus infection. J Gastroenterol Hepatol 1993;8:353357. 8. Sterneck M, Kalinina T, Gunther S, Fischer L, Santantonio T, Greten H, Will H. Functional analysis of HBV genomes from patients with fulminant hepatitis. HEPATOLOGY 1998;28:1390-1397. 9. Locarnini SA, Civitico GM, Newbold JE. Hepatitis B: new approaches for antiviral chemotherapy. Antivir Chem Chemother 1996;7:1-12. 10. Summers J, Smith PM, Horwich AL. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J Virol 1990;64: 2819-2824. 11. Lenhoff RJ, Summers J. Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes. J Virol 1994;68:5706-5713. 12. Lenhoff RJ, Luscombe CA, Summers J. Competition in vivo between a cytopathic variant and a wild-type duck hepatitis B virus. Virology 1998; 251:85-95. 13. Persing DH, Varmus HE, Ganem D. The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid. J Virol 1987;61:16721677. 14. Kuroki K, Russnak R, Ganem D. Novel N-terminal amino acid sequence required for retention of a hepatitis B virus glycoprotein in the endoplasmic reticulum. Mol Cell Biol 1989;9:4459-4466. 15. Mehta A, Lu X, Block TM, Blumberg BS, Dwek RA. Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion. Proc Natl Acad Sci U S A 1997;94: 1822-1827. 16. Hadziyannis S, Raimondo G, Papaioannou C, Anastassakos C, Wong D, Sninsky J, Shafritz D. Expression of pre-S gene-encoded proteins in liver and serum during chronic hepatitis B virus infection in comparison to other markers of active virus replication. J Hepatol 1987;5:253-259. 17. Trautwein C, Schrem H, Tillmann HL, Kubicka S, Walker D, Boker KH, Maschek HJ, et al. Hepatitis B virus mutations in the pre-S genome before and after liver transplantation. HEPATOLOGY 1996;24:482-488. 18. Bock CT, Tillmann HL, Maschek HJ, Manns MP, Trautwein C. A preS mutation isolated from a patient with chronic hepatitis B infection leads to virus retention and misassembly. Gastroenterology 1997;113:19761982. 19. Carman WF, Fagan EA, Hadziyannis S, Karayiannis P, Tassopoulos NC, Williams R, Thomas HC. Association of a precore genomic variant of hepatitis B virus with fulminant hepatitis. HEPATOLOGY 1991;14:219-222. 20. Ogata N, Miller RH, Ishak KG, Purcell RH. The complete nucleotide sequence of a pre-core mutant of hepatitis B virus implicated in fulminant

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32. De Man RA, Bartholomeusz A, Niesters HGM, Zondervan PE, Locarnini S. The sequential occurrence of viral mutations in a liver transplant recipient re-infected with hepatitis B virus: hepatitis B immune globulin escape, famciclovir non-response, followed by lamivudine resistance resulting in graft loss. J Hepatol 1998;29:669-675. 33. Sterneck M, Gunther S, Santantonio T, Fischer L, Broelsch CE, Greten H, Will H. Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation. HEPATOLOGY 1996;24:300-306. 34. Buckwold VE, Xu Z, Chen M, Yen TS, Ou JH. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol 1996;70:5845-5851. 35. Baumert T, Rogers S, Hawegawa J, Liang T. Two core promoter mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J Clin Invest 1996;98:2268-2276. 36. Gunther S, Piwon N, Will H. Wild-type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus with C(1653) 3 T, A(1762) 3 T and G(1764) 3 A mutations in the core promoter. J Gen Virol 1998;79:375-380. 37. Kosalaraksa P, Kavlick MF, Maroun V, Le R, Mitsuya H. Comparative fitness of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 (HIV-1) in an in vitro competitive HIV-1 replication assay. J Virol 1999;73:5356-5363. 38. Imamichi T, Berg SC, Imamichi H, Lopez JC, Metcalf JA, Falloon J, Lane HC. Relative replication fitness of a high-level 3⬘-azido-3⬘-deoxythymidine-resistant variant of human immunodeficiency virus type 1 possessing an amino acid deletion at codon 67 and a novel substitution (Thr 3 Gly) at codon 69. J Virol 2000;74:10958-10964. 39. Delaney WEt, Isom HC. Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus. HEPATOLOGY 1998;28:1134-1146. 40. Delaney WI, Edwards R, Colledge D, Shaw T, Torresi J, Miller TG, Isom HC, et al. Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus. Antimicrob Agents Chemother 2001;45:1705-1713. 41. Melegari M, Scaglioni P, Wands J. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. HEPATOLOGY 1998;27:628-633. 42. Liaw Y-F, Chien R-N, Yeh C-T, Tsai S-L, Chu C-M. Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. HEPATOLOGY 1999;30:567-572. 43. Walter E, Keist R, Niederost B, Pult I, Blum HE. Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. HEPATOLOGY 1996;24:1-5.